Monoclonal antibodies will be prepared from rats immunized with mouse anti-rat cytolytic T lymphocytes (CTLs). The hybridoma-secreted antibodies will be screened for ability to block CTL-mediated killing of a rat lymphoma. The immunization protocol will avoid anti-target (rat lymphoma) antibodies. Antibodies which block (or augment) CTL-mediated killing by binding to CTLs will be used to identify molecules which play crucial functional roles during killing. These antibodies, as well as anti-Lyt-2 and anti-Lyt-3 antibodies known to block killing, will be used to elucidate the molecular basis of killing. SDS-PAGE analysis of antibody-binding CTL molecules will be conducted, initially in collaboration with Dr. Timothy Springer. With regard to function, we will determine at which step each antibody acts: recognition, post-recognition adhesion, or programming for lysis (the lethal hit). We will determine whether the killer target interaction (and which step of the interaction) affects determinant expression on the CTL membrane, or possible shedding or secretion of determinants. We will determine whether (and at which step) CTL determinants are transferred to the target cell membrane. We will determine whether the antibody induces or inhibits triggering of the lethal hit using a new assay we have developed. We will determine whether determinant integrity can be dissociated from function by protease (or glycosidase) treatment of CTLs. In this way, we expect to identify functional CTL molecules and elucidate their modes of action during killing.